Selectin Mediated Cell Adhesion
The migration of white blood cells from the blood to regions of pathogenic exposure in the body is called the inflammatory cascade. Cell adhesion events allow for specific binding of a leukocyte to the endothelium of the vessel that is adjacent to the inflammatory insult; such adhesion events counteract the high vascular shear forces and high blood flow rates that tend to keep the leukocyte circulating, and help guide the leukocyte to the required site.
Four families of vascular adhesion molecules are involved in the migration of leukocytes during the inflammatory response: (1) the integrin family, (2) the counterreceptors of the integrin family, the immunoglobulin superfamily, (3) the selectin family, and (4) the counterreceptors of the selectin family, specialized carbohydrates displayed by the sialomucin adhesion family.
Selectins are also known as "lectin cell adhesion molecules" (LEC-CAMs). Selectins are classified into three groups: L-selectin (LECAM-1, LAM-1, gp90.sup.MEL, Leu-8, TQ-1, CD62L and DREG) is expressed on various leukocytes, and is constitutively expressed on lymphocytes, monocytes, neutrophils, and eosinophils. E-selectin (LECAM-2, CD62E and ELAM-1) is expressed on endothelium activated by inflammatory mediators. P-selectin (GMP-140, PADGEM, LECAM-3 and CD62P) is stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells and is also expressed on endothelium activated by inflammatory stimuli. All members of the selectin family appear to mediate cell adhesion through the recognition of carbohydrates.
The current concept of leukocyte extravasation is based on the consecutive action of several adhesion molecules located on the surface of leukocytes and the endothelium. Lymphocyte extravasation is initiated by the interaction of members of the selectin family and their oligosaccharide-containing counterreceptors. For a review of the current knowledge on lymphocyte adhesion, see e.g., Springer, T. A., Annu. Rev. Physiol. 57: 827-872 (1995).
All selectins bind to sialyl Lewis x (NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc) (sLe.sub.x or sLex) and sialyl Lewis a (NeuNAca2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc) (sLe.sup.a or sLea) as well as related carbohydrate sequences (Bertozzi, C., Chemistry and Biology 2:703-708 (1995)). L-selectin-dependent recognition precedes normal lymphocyte extravasation into peripheral lymph nodes (Gallatin, W. M. et al., Nature 303:30-34 (1983)) and into sites of inflammation (Ley, K. et al., Blood 77:2553-2555 (1991)), both of which are impaired in L-selectin deficient mice (Arbones, M. L. et al., Immunity 1:247-260 (1994)).
Three glycoprotein ligands for L-selectin are currently known: GlyCAM-1, CD34 and MAdCAM-1. The exact structures of the biological ligands of L-selectin are not yet known, but the principal carbohydrate epitopes share some structural features. They are O-glycosidically linked mucin type oligosaccharides with an N-acetyllactosamine backbone, which is 3N-sialylated or 3N-sulphated, 3-fucosylated and sometimes 6- or 6N-sulphated at the distal N-acetyllactosamine termini.
Multivalency of the saccharide ligands enhances selectin binding. Past studies have shown that the ability of an oligosaccharide to inhibit L-selectin-mediated leukocyte adhesion to the endothelium increases with increasing numbers of sialyl L.sup.x groups (Turunen, J. P. et al., J. Exp. Med. 182(4):1133-1141 (1995)), and multivalent sialyl Le.sup.x structures are particularly potent as E-selectin inhibitors (DeFrees, S. A. et al., J. Am. Chem. Soc. 115:7549-7550 (1993); Welply, J. K. et al., Glycobiology 4:259-265 (1994); DeFrees, S. A. et al, J. Am. Chem. Soc. 117:66-79 (1995)). The polylactosamine backbone of P-selectin ligand PSGL-1 is branched and contains several fucoses (Wilkins, P. P. et al., J. Biol. Chem. 271:18732-18742 (1996)), and the presence of multiply fucosylated and multiply sulphated glycans in GlyCAM-1 (Hemmerich, S. et al., J. Biol. Chem. 270:12035-12047 (1995)) suggest that also the single natural carbohydrate ligands for selectins may be multivalent.
High endothelial cells in peripheral lymph nodes express sialyl Lewis a and sialyl Lewis x (sLe.sup.a and sLe.sup.x) epitopes (Paavonen and Renkonen, Am. J. Pathol. 141:1259-1264 (1992); Munro, J. M. et al., Am. J. Pathol. 141:1397-1408 (1992); Sawada, M. et al., Biochem. Biophys. Res. Comm. 193:337-347 (1993)) which are parts of the L-selectin counterreceptor. The endothelial cells in several other locations are sLe.sup.a and sLe.sup.x negative, but inflammatory stimuli can induce previously negative endothelium to express these oligosaccharide structures de novo (Turunen, J. et al., Eur. J. Immunol. 24:1130-1136 (1994)). It has been shown that cultured endothelial cells possess the machinery to generate at least sLe.sup.x, since they have several functional .alpha.2,3 sialyl- and .alpha.1,3 fucosyltransferases, enzymes involved in generating sLe.sup.x from (poly)lactosamines (Majuri, M. et al., Eur. J. Immunol. 24:3205-3210 (1994)).
A number of studies have proposed that selectins are involved in a wide variety of acute and chronic inflammatory conditions in many tissues. It has been proposed that drugs might be designed to impede the deleterious migration of leukocytes that damage tissue in many abnormal inflammatory conditions. However, only very high concentrations (in the mM range) of monomeric charged sugars blocked the adhesion. It has been shown that a specific subset of polyvalent, anionic sugars, such as fucoidin (a polymer of fucose-4-sulfate) and yeast cell wall polyphosphomannan ester (PPME), blocked this adhesion at concentrations in the nM range (Stoolman, L. M. et al., J. Cell Biol. 99:1535-1540 (1984)). In addition, it has been reported that oligosaccharides derived from the sLe.sup.x structure have anti-inflammatory activities. Both the sialic acid-containing (sLe.sup.x) and the sulfate (sulfo-Le.sup.x) forms of this oligosaccharide have been reported to have anti-inflammatory activity in vivo (Lasky, L. A., Annu. Rev. Biochem. 64:113-139 (1995); Mulligan, M. S. et al., Nature 364:149-151 (1993); Mulligan, M. S. et al., J. Exp. Med. 178:623-631 (1993); Buerke, M. et al., J. Clin. Invest. 91:1140-1148 (1994); and Nelson, R. M. et al., J. Clin. Invest. 91:1157-1166 (1993)).
Since lymphocyte infiltration is essential for acute organ transplant rejection (Renkonen, R. et al., Cell. Immunol. 77:188-195 (1983)) analysis of the regulation of lymphocyte traffic into the graft is important. It has been shown that peritubular capillary endothelium (PTCE) in kidney transplants begin to express sLe.sup.x de novo and bind an increased number of lymphocytes during rejection (Turunen, J. et al., Eur. J. Immunol. 24:1130-1136 (1994)).
U.S. Pat. No. 5,352,670 to Venot et al. discloses a method for the enzymatic synthesis of an .alpha.-sialylated oligosaccharide glycoside using sialyltransferase, a CMP-sialic acid analogue as the sialic acid donor and an oligosaccharide glycoside acceptor molecule, having a .beta.Gal(1-3).beta.GlcNAc or .beta.Gal(1-4).beta.GlcNAc disaccharide on the nonreducing terminus.
International Patent Publication No. WO 95/03059 (Gaeta et al.) discloses a synthetic saccharide that contains two glycosidically linked sLe.sup.x moieties, that are useful in blocking cellular adhesion, especially by inhibiting E-selectin binding. These sLex containing oligosaccharides are synthesized on a galactose backbone.